Rho-associated protein kinases (ROCKs), the downstream effectors of Rho A, belong to the member of a serine/threonine protein kinase family. This molecule play key roles in many cellular processes, including the regulation of cell adhesion, migration, contractility, growth, apoptosis, centrosome duplication, and cytokinesis. ROCKs also play the key role in the occurrence of cardiovascular diseases, cancer and neurological diseases. Two ROCK isoforms, have been identified in mammals, ROCKI and ROCKII. They have the similar domain structure and composed of a kinase domain in the N-terminus, a RhoA-binding domain and a pleckstrin homology (PH) domain in the C-terminal region. The signaling pathways for regulating ROCK activity in cells includes: (1) the combination of RhoA and ROCK, (2) proteolytic cleavage of the C-terminal inhibitory domain in apoptotic cells, (3) the intracellular second messengers, such as arachidonic acid, or (4) the interaction with several proteins, such as stimulation by polo-like kinase-1 (Plk1) and nucleophosmin, and repression by GTP binding protein (Gem and Rad, for instance) and the ROCK inhibitor (Morgana).
Recently, the methods for detecting the ROCK activation including: (1) to indirectly determine ROCK phosphorylation by measuring the inhibitor-sensitive phosphorylation ROCK downstream subtracts (e.g. myosin light chain (MLC), myosin binding substrate (MBS), LIM kinase (LIMK), and α-adducin); or (2) to measure the catalytic activity of the purified ROCK kinase by in vitro kinase assay. However, the phosphorylation levels of ROCK downstream subtracts do not directly correspond to the respective activations of ROCKI or ROCKII in cells and tissues. And the ROCK kinase in vitro kinase assay requires high amount of purified ROCK protein that is unfavorable for detecting ROCK activity in cells and tissues. In addition, the loss of RhoA binding during sample preparation might result in the disadvantage of assay. There is no any method can directly and efficiently measure the respective activation of ROCKI and ROCKII. Therefore, these traditional methods are not sufficient to determine ROCKI and ROCKII activation in tissues to evaluate the relationship between the ROCKs activation and diseases such as cardiovascular diseases, cancer or neurological diseases.
It is therefore attempted by the applicant to deal with the above situation encountered in the prior art.